Alice McDonald

An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer

The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin–proteasome system might prove useful for the treatment of human malignancies. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer.

Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells

The reduction of iron is an essential step in the transferrin (Tf) cycle, which is the dominant pathway for iron uptake by red blood cell precursors. A deficiency in iron acquisition by red blood cells leads to hypochromic, microcytic anemia. Using a positional cloning strategy, we identified a gene, six-transmembrane epithelial antigen of the prostate 3 (Steap3), responsible for the iron deficiency anemia in the mouse mutant nm1054. Steap3 is expressed highly in hematopoietic tissues, colocalizes with the Tf cycle endosome and facilitates Tf-bound iron uptake. Steap3 shares homology with F420H2:NADP+ oxidoreductases found in archaea and bacteria, as well as with the yeast FRE family of metalloreductases. Overexpression of Steap3 stimulates the reduction of iron, and mice lacking Steap3 are deficient in erythroid ferrireductase activity. Taken together, these findings indicate that Steap3 is an endosomal ferrireductase required for efficient Tf-dependent iron uptake in erythroid cells.

CCR8 Expression Identifies CD4 Memory T Cells Enriched for FOXP3+ Regulatory and Th2 Effector Lymphocytes

CD4+ Th2 cells are important regulators of allergic inflammation. CCR8 is thought to play a role in Th2-mediated responses, however, expression of CCR8 in peripheral blood has not been fully characterized. Using a fluorescent form of the ligand selective for CCR8 (F-CCL1), we identified the leukocytes expressing CCR8 in human, monkey, and mouse peripheral blood. CCR8 expression is primarily restricted to a subset of human CD4 memory T lymphocytes (15%). Approximately 40% of CCR8+CD4+ T cells express Th2 cytokines IL-4 or IL-13 while 13% express the Th1 cytokine IFN-γ. In fact, 50% of all Th2, but only 5% of Th1, cells express CCR8. Upon anti-CD3/anti-CD28 mAb-mediated activation, CCR8+CD4+ T cells secrete 3- to 7-fold higher levels of IL-4, IL-5, IL-9, and IL-13 and 10- to 20-fold lower levels of IFN-γ or IL-17, compared with CCR8−CD4+ memory T cells. Two-thirds of CCR8+CD4 T cells express cutaneous lymphocyte-associated Ag while the majority lack gut-homing receptors. CCR8+CD4+ cells express CCR7 and CD62L and are present in spleen and lymph nodes of mice. Approximately 25% of CCR8+CD4 T cells express CD25high while 20% of CCR8+CD4+ express the T regulatory cell transcription factor FOXP3 accounting for 60% of all FOXP3-expressing CD4+ T cells. In conclusion, CCR8 marks a diverse subset of CD4 memory T cells enriched for T regulatory and Th2 cells which have the potential for recruitment into sites of allergic inflammation where they could participate in the induction and regulation of the allergic response.

MLN8054, an Inhibitor of Aurora A Kinase, Induces Senescence in Human Tumor Cells Both In vitro and In vivo

Aurora A kinase is a serine/threonine protein kinase responsible for regulating several mitotic processes including centrosome separation, spindle assembly, and chromosome segregation. Small molecule inhibitors of Aurora A kinase are being pursued as novel anticancer agents, some of which have entered clinical trials. Despite the progress in developing these agents, terminal outcomes associated with Aurora A inhibition are not fully understood. Although evidence exists that Aurora A inhibition leads to apoptosis, other therapeutically relevant cell fates have not been reported. Here, we used the small molecule inhibitor MLN8054 to show that inhibition of Aurora A induces tumor cell senescence both in vitro and in vivo. Treatment of human tumor cells grown in culture with MLN8054 showed a number of morphologic and biochemical changes associated with senescence. These include increased staining of senescence-associated β-galactosidase, increased nuclear and cell body size, vacuolated cellular morphology, upregulation/stabilization of p53, p21, and hypophosphorylated pRb. To determine if Aurora A inhibition induces senescence in vivo, HCT-116 xenograft–bearing animals were dosed orally with MLN8054 for 3 weeks. In the MLN8054-treated animals, increased senescence-associated β-galactosidase activity was detected in tissue sections starting on day 15. In addition, DNA and tubulin staining of tumor tissue showed a significant increase in nuclear and cell body area, consistent with a senescent phenotype. Taken together, this data shows that senescence is a terminal outcome of Aurora A inhibition and supports the evaluation of senescence biomarkers in clinic samples. Mol Cancer Res; 8(3); 373–84

Treatment-Emergent Mutations in NAEβ Confer Resistance to the NEDD8-Activating Enzyme Inhibitor MLN4924

MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE) in clinical trials for the treatment of cancer. MLN4924 is a mechanism-based inhibitor, with enzyme inhibition occurring through the formation of a tight-binding NEDD8-MLN4924 adduct. In cell and xenograft models of cancer, we identified treatment-emergent heterozygous mutations in the adenosine triphosphate binding pocket and NEDD8-binding cleft of NAEβ as the primary mechanism of resistance to MLN4924. Biochemical analyses of NAEβ mutants revealed slower rates of adduct formation and reduced adduct affinity for the mutant enzymes. A compound with tighter binding properties was able to potently inhibit mutant enzymes in cells. These data provide rationales for patient selection and the development of next-generation NAE inhibitors designed to overcome treatment-emergent NAEβ mutations.

Potential biomarkers of bortezomib activity in mantle cell lymphoma from the phase 2 PINNACLE trial

Immunohistochemical analyses of archival tumor specimens were used for pre-planned exploratory analyses of protocol-specified candidate biomarkers of bortezomib activity in 73 patients with relapsed/refractory mantle cell lymphoma in the phase 2 PINNACLE study. Consistent with other studies, elevated Ki-67 was a marker of poor prognosis, demonstrating significant associations with shorter time to progression and overall survival. Elevated NF-κB p65 and low PSMA5 expression demonstrated a trend for better response and were significantly associated with longer time to progression; elevated NF-κB p65 demonstrated a trend toward longer overall survival. This is consistent with myeloma clinical genomics research, suggesting biomarker relevance across tumor types. Elevated p27 was significantly associated with longer overall survival. Overall survival analyses by International Prognostic Index and Mantle Cell Lymphoma International Prognostic Index confirmed differential prognosis by both scores. These biomarkers data begin to illuminate bortezomibs mechanism of action in lymphoma.

Tazemetostat, an EZH2 inhibitor, in relapsed or refractory B-cell non-Hodgkin lymphoma and advanced solid tumours: a first-in-human, open-label, phase 1 study

BACKGROUND Activating enhancer of zeste homolog 2 (EZH2) mutations or aberrations of the switch/sucrose non-fermentable (SWI/SNF) complex (eg, mutations or deletions of the subunits INI1 or SMARCA4) can lead to aberrant histone methylation, oncogenic transformation, and a proliferative dependency on EZH2 activity. In this first-in-human study, we aimed to investigate the safety, clinical activity, pharmacokinetics, and pharmacodynamics of tazemetostat, a first-in-class selective inhibitor of EZH2. METHODS We did an open-label, multicentre, dose-escalation, phase 1 study using a 3 + 3 design with planned cohort expansion at the two highest doses below the maximally tolerated dose. The study was done at two centres in France: Institut Gustave Roussy (Villejuif, Val de Marne) and Institut Bergonié (Bordeaux, Gironde). Eligible patients had relapsed or refractory B-cell non-Hodgkin lymphoma or an advanced solid tumour and were older than 18 years, with Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate end-organ function. Tazemetostat was administered orally from 100 mg twice daily to 1600 mg twice daily in 28-day cycles. The primary endpoint was to establish the maximum tolerated dose or recommended phase 2 dose of tazemetostat, as determined by dose-limiting toxicities, laboratory values, and other safety or pharmacokinetic measures in cycle one according to local investigator assessment. Safety was assessed in patients who received at least one dose of tazemetostat; antitumour activity was assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01897571. The phase 1 part of the study is complete, and phase 2 is ongoing. FINDINGS Between June 13, 2013, and Sept 21, 2016, 64 patients (21 with B-cell non-Hodgkin lymphoma, and 43 with advanced solid tumours) received doses of tazemetostat. The most common treatment-related adverse events, regardless of attribution, were asthenia (21 [33%] of 64 treatment-related events), anaemia (nine [14%]), anorexia (four [6%]), muscle spasms (nine [14%]), nausea (13 [20%]), and vomiting (six [9%]), usually grade 1 or 2 in severity. A single dose-limiting toxicity of grade 4 thrombocytopenia was identified at the highest dose of 1600 mg twice daily. No treatment-related deaths occurred; seven (11%) patients had non-treatment-related deaths (one at 200 mg twice daily, four at 400 mg twice daily, and two at 1600 mg twice daily). The recommended phase 2 dose was determined to be 800 mg twice daily. Durable objective responses, including complete responses, were observed in eight (38%) of 21 patients with B-cell non-Hodgkin lymphoma and two (5%) of 43 patients with solid tumours. INTERPRETATION Tazemetostat showed a favourable safety profile and antitumour activity in patients with refractory B-cell non-Hodgkin lymphoma and advanced solid tumours, including epithelioid sarcoma. Further clinical investigation of tazemetostat monotherapy is ongoing in phase 2 studies in adults and a phase 1 study for children, which are currently enrolling patients who have B-cell non-Hodgkin lymphoma and INI1-negative or SMARCA4-negative tumours. FUNDING Epizyme and Eisai.

PRELIMINARY EVIDENCE OF A MOLECULAR PREDICTOR OF TAZEMETOSTAT RESPONSE, BEYOND EZH2 MUTATION, IN NHL PATIENTS VIA CHARACTERIZATION OF ARCHIVE TUMOR AND CIRCULATING TUMOR DNA

Introduction: EZH2, a histone methyl transferase subunit of Polycomb repressor complex 2, is frequently mutated in DLBL. Inhibitors of EZH2 have demonstrated promising responses in early clinical trials. We examined the frequency of EZH2 mutation in 2 large prospective series of DLBL and correlated this to clinical outcomes in relation to other biological features. Methods: Patients (pts) received standard immunochemotherapy regimens as first‐line treatment for DLBL. Sanger sequencing (SS) focusing on “hotspot” mutation sites in exons 16 and 18 was successful in 1052 of 1097 DLBL samples enrolled in the UK NCRI Molecular Profiling for Lymphoma (MaPLe) study. Next generation sequencing (NGS) using Fluidigm Access Array PCR and Illumina MiSeq was used to profile a separate cohort of 365 pts enrolled in the UK NCRI/ SAKK REMoDL‐B trial (NCT01324596) (CRUKE/10/024). In these cases, cell of origin (COO) was determined by gene expression profiling (GEP) using Illumina WG‐DASL. Results: EZH2 mutations were detected in 9% of DLBL pts (98/1052) by SS and 15% (54/365) by NGS. Ninety‐five percent of mutations were at Y646 position in exon 16. EZH2mutations were strongly associated with GCB subtype, occurring in 27% of cases (50/185) versus 0/ 106 in ABC subtype and (4/71) in unclassified subtype (P < .0001). Overall, EZH2 mutations were not significantly associated with age, sex, performance status, stage, or IPI, compared to unmutated GCB DLBL. PFS was similar between EZH2 mutated and unmutated GCB DLBL subtype: 78.5% vs 80.7% at 30 months, HR 1.06 (95% CI, 0.62‐1.81) (P = .844). A subset of GCB cases showed Burkitt‐like GEP, associated with inferior progression free survival (PFS) HR 2.21 (95% CI, 1.28‐7.73) (P = .012), among which 11/24, where mutation status was available, had EZH2 mutations. There was heterogeneity in progression free survival identified by presence or absence of EZH2 mutations and Burkitt‐like gene expression signature. Conclusions: EZH2 mutations are significantly associated with the DLBL GCB‐subtype and more common in cases identified as Burkitt‐ like by GEP. Overall outcomes are similar in mutant and wild‐type cases when adjusted for COO and IPI, but Burkitt‐like cases that carry EZH2 mutations may be a preferential subset in which to test targeted therapies.

Abstract C12: Identification of biomarkers and pathways associated with response to the DOT1L inhibitor Pinometostat (EPZ-5676) in MLL-r leukemia

Pinometostat is a highly selective first in class DOT1L inhibitor currently in Phase 1 clinical trials in adult and pediatric leukemia patients (pts) with MLL rearrangements (MLL-r or MLL-PTD). Preliminary results of the adult trial have demonstrated clinical activity including complete remissions in a subset of patients (Stein, 2014). Investigation and identification of candidate molecular correlates of pinometostat response in both pt samples and cell lines are reported. RNA and DNA were isolated from PBMCs and/or bone marrow collected prior to treatment from 18 pts enrolled in the adult pinometostat Phase 1 study (CT.gov: NCT01684150), at the following doses 24 (n = 2), 36 (n = 3), 54 (n = 6), 80 (n = 3) and 90 mg/m2/day doses (n = 4). mRNA transcript abundance was assessed using whole genome RNASeq and DNA variants were determined using a 194 gene panel, MyAML (Genection Inc.). Correlations of transcript abundance and DNA variants detected with categorical (responder = CR or PR [n = 3], or no response [n = 16]) and continuous response parameters (time on study [TOS], mean = 59 days: range = 8-196 days) were performed. For cell lines, whole genome RNASeq data was generated from 14 cell lines (MLL-r or MLL-PTD) with a range of in vitro sensitivity to pinometostat (cell proliferation IC50 2 nM to > 10 μM) and RNA transcripts identified as correlated with IC50 were submitted for pathway analysis. Univariate analyses revealed no DNA variants to be associated with either response category (FDR adjusted P MLL-r fusion partner (e.g. t(11:19)) may influence clinical response to pinometostat. In addition RNASeq based characterization of patient samples and cell lines revealed candidate pathways that may cooperate with or antagonize pinometostat activity that warrant further investigation. Citation Format: Scott Daigle, Alice McDonald, Ty M. Thomson, David A. Drubin, Michael Maria, A. Carson, Brad Patay, Jeff Keats, Christine Klaus, Alejandra Raimondi, G. Garcia-Manero, D. A. Rizzieri, Raoul Tibes, Jesus Berdeja, Eytan M. Stein, Blythe Thomson, Stephen J. Blakemore. Identification of biomarkers and pathways associated with response to the DOT1L inhibitor Pinometostat (EPZ-5676) in MLL-r leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C12.

Tissue microarray analysis reveals protein expression patterns and potential biomarkers of clinical benefit to bortezomib in relapsed/refractory non‐Hodgkin lymphoma

Patients with mantle cell lymphoma (MCL) and follicular lymphoma treated with bortezomib have consistent response rates of 30–50% across several clinical trials, suggesting a common tumour biology that may predict response. It remains unknown which processes affected by proteasome inhibitors (PI) are most important in their activity in nonHodgkin Lymphoma (NHL). Leading theories include inhibition of the cell cycle, nuclear factor jB (NFKB1; NF-jB) signalling, angiogenesis and decreased degradation of antiapoptotic proteins. Between June 2001 and December 2006, 103 patients enrolled in a multicentre Cancer Therapy Evaluation Program-sponsored Phase II trial and were treated with singleagent bortezomib. Eligibility criteria included: (i) confirmed indolent NHL or MCL, (ii) <3 prior cytotoxic treatment regimens, and (iii) adequate organ reserve (O’Connor et al, 2005; Gerecitano et al, 2009). Tissue microarrays (TMA) were stained for a panel of targets selected for their possible prognostic associations with NHL or mechanisms of action (MOA) of PI (Table S1). We correlated outcome with pretreatment tumour protein expression patterns in this unique patient population. Tissue microarrays were constructed from 55 pre-treatment tissue blocks as previously published (Koreishi, 2010). Stains for CDKN1A (p21), CDKN1B (p27), BIRC5 (Survivin), TP53 (p53), BCL2, MCL1, CFLAR (c-FLIP), Caspase, VCAM1, REL/RELA (p65), PSMB1 (proteasome subunit b1), PSMA5 (proteasome subunit a5), TOP2A(TOPOa), MIB1/ MKI67 (Ki67), CCND1 (cyclin D1), MUM1, BCL6 and CTAG1B (NYEso) were graded by two independent pathologists, and a consensus grade was determined for each marker. Cases were considered positive if >20% of cells showed staining. In cases of discrepancy, the higher value was reported. Stains were also graded on a scale from 0 to 4, representing the proportion of cells per high power field. Nuclear RELA was estimated using continuous percentages, and then separated into quartiles for analysis. In those cases where the diagnostic samples did not run through the entire TMA block, shavings from the initial block were graded separately. Fisher’s exact test was used to assess the association between response rates [progressive disease (PD) versus complete response (CR)/partial response (PR)/stable disease (SD), CR/PR versus SD/PD] and the expression of each marker. Progression-free survival (PFS) was defined from the start of treatment to the date of death or progression, whichever occurred first. Patients were censored at their last date of follow-up if they were alive and progression-free. For patients who responded to therapy (CR/PR), duration of response (DOR) was calculated from the date of the best response to the date of progression, and patients who remained progression-free at the last follow-up were censored. None of the patients included in this analysis died prior to disease progression. The Log-rank test or permutation log-rank test was used to compare PFS and DOR between levels of stain expression. Multiple comparison adjustment was not applied considering the exploratory nature of the study. All tests were completed in SAS 9.2 (SAS Institute, Inc., Cary, NC, USA) and R version 2.9.2 (http:// www.r-project.org/). Demographic and response/survival information for patients included in this analysis (n = 55) reflect the total population (n = 103, Table 1) (Gerecitano et al, 2009; O’Connor et al, 2010). Two proteins showed significant association with response: High expression (50–75%) of MCL1 conferred a decreased chance of achieving SD or better compared with 25–50% or 0–25% expression of MCL (54%, 100% and 73%, respec-